Membrane Targeting of a Rab GTPase That Fails to Associate with Rab Escort Protein (REP) or Guanine Nucleotide Dissociation Inhibitor (GDI)*

Abstract

The targeting of various Rab proteins to different subcellular compartments appears to be determined by variable amino acid sequences located upstream from geranylgeranylated cysteine residues in the C-terminal tail. All nascent Rab proteins are prenylated by geranylgeranyltransferase II, which recognizes the Rab substrate only when it is bound to Rab escort protein (REP). After prenylation, REP remains associated with the modified Rab until it is delivered to the appropriate subcellular membrane. It remains unclear whether docking of the Rab with the correct membrane is solely a function of features contained within the prenylated Rab itself (with REP serving as a "passive" carrier) or whether REP actively participates in the targeting process. To address this issue, we took advantage of a mutation in the ␣2 helix of Rab1B (i.e. Y78D) that abolishes REP and GDI interaction without disrupting nucleotide binding or hydrolysis. These studies demonstrate that replacing the C-terminal GGCC residues of Rab1B(Y78D) with a CLLL motif permits this protein to be prenylated by geranylgeranyltransferase I but not II both in cell-free enzyme assays and in transfected cells. Subcellular fractionation and immunofluorescence studies reveal that the prenylated Rab1B(Y78D)CLLL, which remains deficient in REP and GDI association is, nonetheless, delivered to the Golgi and endoplasmic reticulum (ER) membranes. When the dominant-negative S22N mutation was inserted into Rab1B-CLLL, the resulting monoprenylated construct suppressed ER 3 Golgi protein transport. However, when the Y78D mutation was added to the latter construct, its inhibitory effect on protein trafficking was lost despite the fact that it was localized to the ER/Golgi membrane. Therefore, protein interactions mediated by the ␣2 helical domain of Rab1B(S22N) appear to be essential for its functional interaction with components of the ER 3 Golgi transport machinery.

Dual Prenylation Is Required for Rab Protein Localization and Function

“…ypt1 CIIL and sec4 CIIL , the substrates for either GGTaseI or GGTaseII, were able to suppress temperaturesensitive alleles ypt1-3 and sec4-8, while the exclusive GGTaseII substrates ypt1 C205S and sec4 C214S were not. These data agree with previous studies demonstrating that Rab proteins mutated to GGTaseI substrate CAAX boxes can in fact support function, provided that sufficient Rab protein reaches the correct membrane (Soldati et al, 1993;Overmeyer et al, 2001). It is possible that the ypt1 C205S and sec4…”

Role of Rab GTPases in Membrane Traffic and Cell Physiology

“…Chimeras of Rab1 or Rab5 with the hypervariable region of Rab9 (that interacts with TIP47) can be relocated from the Golgi (normal Rab1 localization) or the early endosome (normal Rab5 localization) to the late endosome (normal Rab9 localization) upon overexpression of TIP47 (1). More recent studies in mammalian cells show that certain F and SF regions of Rabs are more important than their hypervariable domains for membrane targeting and implicate interactions with effector proteins for proper localization (3, 307). It is important to note that the hypervariable region contains a motif that interacts with proteins that regulate the membrane-bound state of the Rab (see below).…”

Identification and Characterisation of the RalA-ERp57 Interaction: Evidence for GDI Activity of ERp57

“…We therefore designed point mutations in RalA’s switch II region with the aim of identifying important amino-acid residues in RalA for ERp57 binding. The mutations D74A, Y75A, A77R and I78N were selected based on a modelled structure of RalA and a knowledge of previous mutations affecting the GDP-dependent interactions of other small GTPases [5], [41], [42]. The mutation D49A (from switch I) in RalA was included as a control that was previously reported to selectively lose interaction with RalBP1 and not the exocyst [18], [43].…”